Capillary Electrophoresis-electrospray Ionization-mass Spectrometry Research Articles

Simultaneous measurement of nineteen binding constants of peptides to vancomycin using affinity capillary electrophoresis-mass spectrometry.

On-line affinity capillary electrophoresis-electrospray ionization-mass spectrometry (ACE-MS) was used for the simultaneous measurement of multiple binding constants of an all-D-tetrapeptide library to the model receptor, vancomycin. Determination of Kd values for the 19 peptides of the form Fmoc-DXYA is demonstrated. The data are compared with the results obtained for individual compounds using ACE-UV, and good correlation between the two detection methods is shown. Simultaneous determination of multiple Kd values by ACE-MS is achieved in one set of experiments, whereas only one Kd value can be obtained by ACE-UV during the same time. ACE-MS measures multiple binding constants in solution in a fast and reliable manner using femtomole amounts of samples.

A New High-performance Interface for Capillary Electrophoresis/Electrospray Ionization Mass Spectrometry

A novel capillary electrophoresis electrospray ionization mass spectrometry (CE/ESI-MS) interface has been developed based upon an alternative means of establishing electrical contact at the CE capillary terminus. In this design, the terminus of the CE separation capillary and a short ESI emitter capillary are connected and sheathed by polysulphone micro-dialysis tubing. The electrical connection, to close the CE circuit and simultaneously establish the electrospray voltage, is provided outside the membrane via an electrode in a small liquid reservoir. In contrast to the sheath flow and liquid junction interfaces, the flow rate of liquid to the ESI source consists of the CE eluent and is not increased. The use of a "micro-spray' type emitter produces a stable electrospray at the very low flow rates typical of CE, providing a highly sensitive, efficient interface. Post-separation acidification of analytes via the dialysis junction is also demonstrated.

Determination of amino acids by on-line capillary electrophoresis-electrospray ionization mass spectrometry.

On-line capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESMS) has been used for the separation and detection of amino acid mixtures. Four natural amino acids, histidine, tryptophan, phenylalanine, aspartic acid, and a tripeptide, glutathione, were separated and determined. Protonated molecules were detected in the CE-ESMS mode with detection limits of about one pmol. Optimum CE-ESMS operating conditions for amino acid analysis were determined employing acetic acid solutions as CE electrolytes. The examined parameters included capillary diameter (50-100 microns internal diameter), applied separation voltage (20-30 kV), and concentration of electrolyte (10-60% acetic acid). Stable working conditions were maintained when the CE currents were less than 18 microA. The use of electrolyte solutions such as those described here instead of true buffer solutions may have advantages for CE-ESMS systems which employ a "sheathless" interface.

Fumonisin B1 analysis with capillary electrophoresis-electrospray ionization mass spectrometry.

Fumonisin B1, a mycotoxin produced by a common fungal contaminant of corn, Fusarium moniliforme, was analyzed by capillary electrophoresis-electrospray ionization mass spectrometry. System performance was maximal with uncoated columns. System efficiencies of approximately 44,000 plates/m and reproducible analysis times of about 13 min were obtained. System efficiency with methyl-coated columns was approximately 24,000 plates/m. Reproducible analysis times of about 3.5 min were obtained with these columns. With uncoated columns, the concentration limit of detection was 156 ppb with a s/n ratio of approximately 10. The estimated injected mass at 156 ppb was 1.1 pg. Repeated injections of extracts containing constant fumonisin B1 concentrations showed that peak areas were slightly inconsistent, although generally similar to variations encountered with liquid chromatography-electrospray ionization mass spectrometry. The source of this inconsistency was traced to sample solubility, errors inherent in electrophoresis injections, and electrospray instability. Minimizing these problem areas will produce a technique with peak area reproducibilities comparable to liquid chromatography-electrospray ionization mass spectrometry, but with potentially greater resolving power.

A study of the thermal denaturation of ribonuclease S by electrospray ionization mass spectrometry

The thermal stability of ribonuclease S (RNase S), and enzymatically active noncovalent complex composed of a 2166-u peptide (S-peptide) and a 11,534-u protein (S-protein), was investigated by electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis ESI-MS (CE-ESI-MS). The intensities of peaks corresponding to the RNase S complex were inversely related to both the applied nozzle—skimmer (or capillary—skimmer) voltage bias in the atmosphere—vacuum interface and the temperature of the RNase S solution. By using a heated metal capillary—skimmer interface and a room temperature solution of RNase S, the intensities of RNase S molecular ion peaks were observed to decrease with increasing metal capillary temperature. Mass spectrometric studies with both the nozzle—skimmer and capillary—skimmer interface designs allowed determination of phenomenological enthalpies for dissociation of the RNase S complex in both solution and for the electrosprayed microdroplet—gas phase species. Intact RNase S complex could also be detected with CE-ESI-MS separations by using a 10-mM ammonium bicarbonate (pH 7.9) solution as the electrophoretic buffer. These studies provide new insights into the stability of multiply charged noncovalent complexes in the gas phase and the mass spectrometric conditions required for such studies, and suggest that information regarding solution properties can be obtained by ESI-MS.

Investigation of the metabolic fate of the neuroleptic drug haloperidol by capillary electrophoresis-electrospray ionization mass spectrometry

Combined capillary electrophoresis—mass spectrometry (CE—MS) is a powerful analytical tool for the characterization of components of complex mixtures. Furthermore, when interfaced with an electrospray ionization (ESI) source, skimmer induced fragmentation can yield structural information that aids compound identification. We show that these techniques can be used to probe the metabolic fate of drugs by investigation of the phase I metabolism of the widely used neuroleptic drug haloperidol. In addition, we demonstrate differences of in vitro metabolism of haloperidol by mouse and guinea pig hepatic microsomes.

Sensitivity considerations for large molecule detection by capillary electrophoresis-electrospray ionization mass spectrometry

The use of the electrospray ionization (ESI) method for interfacing capillary electrophoresis with mass spectrometry (CE-MS) is particularly well suited for the analysis of large molecules due to the multiple charging phenomenon. While ionization efficiency is very high, the available ion current is dispersed over more peaks so that the maximum peak intensity obtainable declines significantly for large molecules. Sensitivity with ESI can be improved by operation at very low flow-rates, an ideal situation for CE-MS. These and other considerations related to sensitivity are illustrated using ESI-MS measurements for cytochrome c.